Study Of The Distribution And Property Of The Nerve Fibers In Bone

[ Backgrand ]One of the major problem is how to construct and survivalthe tissue engineering bone of the large volume and produce a marked effect, along with developing research of tissue engineering and transition to clinic application. The compound tissue engineering bone with the innervation and blood supply is one of the desired plan, and it correspond the bionics. The blood supply of bone had detailed research and the vascularized tissue engineering bone have practicable approach. But, the knowledge about the innervation of bone is so poor in the point of view of anatomy and physiology. So it is necessary to explore the innervation of the normal bone. The problem we are currently faced with is how to morphological distributation and character of the nerval fiber is in the long bone and whether we need to neuralizate the tissue engineering bone.Where does come from the nerve in bone? Is it sensory nerve, motor nerve or sympathetic nerve? This question had disputed for many years. The coincidence is to exist the sympathetic nerve in long bone. In 19th century, the researcher had observed the innervation is necessary to development and regeneration in amputated extremity. For rodents and advanced mammal the major research demonstrate the denervation of operating or chemical approach can make the change in bone mineral deposition and osteal structure. But it can't alter the development in bone. The brain trauma canstimulate callus formation and union of fracture in bone. There are some experimental results indicate denervation can descend growth rate and delay osseous maturation. It is demonstrated the nerve participate the development of bone. In order to demonstrate the efferent sympathetic fiber can regulate function of marrow, it is observed that the electric stimulation of sympathetic nerve cause vasoconstriction and increase of erythrocyte, leucocyte and normoblast in blood circulation. The innervation of the efferent sympathetic fiber in marrow is confirmed by this experiment.The neuropeptide is one of the bioactivity polypeptide discovered in 30's of 20th. The research increase about it along with the utilization in technique of radioimmunity, cytochemistry and molecular biology. The immunohistochemistry provid a new approach to research the innervation of bone. The peptidergic nerve had been discovered in periosteum, compact bone, cancellous bone and bone marrow. Now it is realized varied neuropeptide the bone, so as CGRP, NPY, SP,VIP and TH. But the function in bone metabolism and distribution of that need to been research further.It is demanded to solve the ploblem of the innervation of bone in the detailed morphological characteristic and that the nerve in bone come from somatic nervous system or autonomic nervous system and whether nutrient foramen is the ingress of the nerve in long bone and how many diameter the nerve is in nutrient foramen and which peptidergic nerves are composed in bone and what the neuropeptide effect the metabolism in bone?[Objective]To observe the distribution of the nerval fiber in bone and its detailed morphological characteristic in compact bone, cancellous bone and bone marrow. To comprehend the relative in innervation and blood supply. To analyze the relative infunction and morphological characteristic which the nerve distribute in the vicinity of osteocyte, osteoblast, osteoclast and myeloid element. To understand the construction features of the nerval fiber in bone in ultrastructure. To measure diameter of nerve fiber in bone, and to affirm the ingress of the nerve in long bone.To affirm the composition of the nerve fiber in bone, the sensory, motor or sympathetic nerve. To identify the relative in bone nerve and central nerve and the distribution of neurotransmitter.To observe the distribution of different neuropeptide CGRP, NPY and TH in bone and analyze the characteristic. To discuss the mechanism and effect of the neuropeptide on bone metabolism in point of cytobiologic view.To observe microstructure of bone and identify the morphologic relative and characteristic of bone canaliculi, bone lacuna and Haversian canal. To analyze the three-dimensional structure and passageway of nutritional transmission in bone.[Method]1. The nerve fiber in bone and the microstructure of boneThe polished bone: To make the tibial example from fresh cadaver in metaphysic and diaphysis and epiphysis respectively. To polish the bone and silver impregnation. To observe the specimen under the light microscope and confocal microscopy and find the nerve fiber.To make the tibial example from fresh cadaver and find the nutrient foramen along blood vessel. To saw the bone into 2~3mm thin slice in the center of nutrient foramen and decalcificate in EDTA. To make slice in HE staining and silver impregnation respectively. To observe the specimen under the light microscope and find the nerve fiber.To make the tibial example from fresh cadaver in bone marrow, cancellous bone, compact bone, metaphysic, diaphysis and epiphysis respectively. To decalcificate2-14 days in EDTA. To make slice in HE staining and silver impregnation respectively. To observe the specimen under the light microscope and find the nerve fiber.To make the tibial example from fresh cadaver of neonatus and adult in bone marrow, cancellous bone, compact bone, metaphysic, diaphysis and epiphysis respectively. To decalcificate 1-7 days in EDTA. To make sample of transmission electron microscope and observe it.To make the tibial example from fresh cadaver of neonatus and adult in bone marrow, cancellous bone, compact bone, metaphysic, diaphysis and epiphysis respectively. To make sample of scanning electron microscope and observe it.To make the tibial example from fresh cadaver in compact bone along cross section and longitudinal section respectively. To make sample of atomic force microscope and observe it.2. Autoradiographic nerve tracing methodThe Newzealand rabbits had been divided 4 group and 4 per group. To inject the JH-Leucine of 3mlx25mci/ml into the bone marrow of femoral bone in every rabbits. To execute the rabbits in 3, 5, 7 and 10 day post operation of every group respectively and obtain sample of the L2 section spinal cord and postmesentery ganglion and soft tissue of vicinal postmesentery ganglion. The sample had been treated respectively: 1. the quantitation of 3H radiation and statistics analysis. 2. radioautography. To slice sample into thickness of lOum and make radioautography.3. peptidergic nerve in boneTo make the tibial example from fresh cadaver of neonatus and adult in bone marrow, cancellous bone, compact bone, metaphysic, diaphysis and epiphysis respectively. To decalcificate in EDTA. To stain the sample in immunohistochemistry, and the antibody is CGRP, NPY and TH. To identify positive location and analyzethe positive in different cells. [Result]1 The nerve fiber in bone and the microstructure of bone 1. 1 The observation under light microscope and confocal microscopy Compact bone: The nerve fibers enter the Haversian system along the blood vessel in bone. Its origin is outside of bone. The nerve fibers get into the bone marrow through Haversian canal and Volkmann's canal and its diameter is 3~8um in Haversian canal. The distribution of nerve fibers in bone matrix and how to contact the osteocyte can't been observed. Many nerve fibers are observed in periosteum, and some of that permeate the muscle and get into subperiosteal. In bone trabecular and bone marrow there are many nerve fibers endings which arrange around the blood vessel. Some of nerve fibers are diffused distribution and observed in vicinity of hematopoietic cell. There are sporadic nerve endings in epiphyseal plate and no nerve fibers permeate epiphysis to diaphysis. There are no distribution of nerve fibers in cartilaginous part.1.2 The structure of nutrient foramenThe nerve fibers passing nutrient foramen are observed and following the blood vessel. The diameter of nutrient foramen is 0.5-1.0mm, and the diameter of nerve fiber, arteriole and venula in nutrient foramen is 7~15u,m, 3O~8Oum and 60~200um respectively.1.3 The observation in transmission electron microscope and scanning electron microscope.Transmission electron microscope: To observe that the nerve fibers appear in vicinity of osteocyte in cancellous bone. There are characteristic of few vacuole, many microfilament and micro-tubular structure,sparing mitochondria and rough endoplasmic reticulum, free ribosome and vesicle structure of cellular membrane inthe nerve cell. The Schwann cells had been find in vicinity of osteocytes. Some of nerve fibers appear along vascular endothelial cell or between vascular endothelial cell and osteocytes or between osteocyte and membrane of matrix cell. There are no classic synaptic structure. Scanning electron microscope: the Haversian canal distinctly arrange in bone and the collagen fibers regularly appear and the crystal is circular permutation.1.4 The observation in atomic force microscopeTo observe the distribution of nerve fiber in bone. The crystal of Ca-P are columnar arrangement, and the bone canaliculi link up the bone lacuna and Haversian canal. The diameter of bone canalicul is 100~300nm, average 200.2±51.8nm. The bone lacuna is elliptic, and its long axis diameter is 4.1~10.5um, average 7.41 + 1.82um. There are 20-40 bone canalicul with every bone lacuna. The bone lacuna arrange regularly around Haversian canal. The interval between bone lacuna is 10~40um. the size of crystal is 80~20Onm, average 174.6 ±57.5nm.2 Autoradiographic nerve tracing methodIt is all positive to detect 3H in spinal cord and sympathetic ganglia. After 7 and 5 day in operation, it reach the peak in spinal cord and sympathetic ganglia respectively. In the Autoradiography, the positive sign concentrate the cornu posterius medullae spinalis. The outline of sympathetic ganglia are obviously observed.3 The neuropeptide in boneThere are a great quantity of CGRP, NPY and TH immunoreactive fibers in bone. The CGRP immunoreactive fibers principally distribute the region of activated metabolism. In the epiphyseal growth plate and periosteum, it is strong expression. In vicinity of the osteocyte and hematopoietic cell in cancellous bone and bone marrow, it is medium expression. In surrounding blood vessel of Haversian canal and bone marrow, it is faint expression. The NPY immunoreactive fibers principally distributethe region of surrounding blood vessel. In vicinity of blood vessel in epiphyseal growth plate, it is strong expression. In Haversian canal and bone marrow, it is it is medium expression. In vicinity of the osteocyte and hematopoietic cell in cancellous bone and bone marrow, it is faint expression. The TH immunoreactive fibers principally distribute the region of surrounding blood vessel in bone marrow and epiphyseal growth plate. In vicinity of blood vessel in bone marrow and epiphyseal growth plate, it is strong expression. In vicinity of hematopoietic cell of bone marrow, it is medium expression. In osteocyte and chondrocyte in epiphyseal growth plate, it is faint expression.[ Conclusion ]There are a great quantity of nerve fibers in human long bone. The nerve fibers along with blood vessel arrange in Haversian canal. Its diameter is 3~8um in Haversian canal. The nerve fibers observed is medullated nerve fibers. The majority of nerve fibers accompany blood vessel in cancellous bone and bone marrow. A few of nerve fibers are diffused distribution. The ending of nerve fibers are observed. The nerve fibers can directly regulate the vasoconstriction and the osteocyte's metabolism. The classic synaptic structure hadn't been observed.The nutrient foramen is way of the nerve fibers into bone tissue, with companying blood vessel. The diameter of nerve fibers in nutrient canal is 7~15um. The nerve fibers observed in nutrient canal is medullated nerve fibers.There are sympathetic nerve and sensory nerve in bone.There are a great quantity of peptidergic nerve. The CGRP immunoreactive fibers principally distribute the region of activated metabolism in bone. The function of CGRP in bone is to regulate the metabolism of osteocyte. There are two way that CGRP effect metabolism of osteocyte. One is directly regulate the osteoblast and osteoclast. Another is the regulate vasoconstriction to effect the metabolism ofosteocyte indirectly. The NPY and TH immunoreactive fibers principally distribute the region of surrounding blood vessel and their function is regulation of asoconstriction to effect the metabolism of osteocyte indirectly.The bone tissue is complicated structure. There are integrated lacunar system in it. The nutrient foramen is passage connecting outside. The nerve fibers and blood vessel go straight through it. The Volkmann's canal link up between Haversian canal. In the osteon, the osteocyte exist in bone lacuna and the bone canaliculi is the passage of exchanging nutrient substance and molecular signal into osteocyte.